The present invention relates to the use of recombinant DNA technology for in vivo gene transfer using implanted retroviral producer cells. Specifically, the invention relates to the therapy of brain tumors using modified producer cells to make brain tumor cells sensitive to chemotherapeutic agents.
Brain tumors are major causes of morbidity and mortality, particularly among young people. Moreover, their incidence appears to be increasing for unknown reasons. The causes of brain tumors are not known, although radiation, pollutants, and electromagnetic fields are suspected. Most brain tumors are inoperable; even for those brain tumors that are operable, operations to remove them are extremely difficult and delicate and frequently leave neurological deficits. There is a need for more efficient chemotherapeutic treatment of brain tumors.
One possible avenue of treatment for brain tumors, as yet little explored, involves intracerebral neural grafting of cells that produce anti-cancer agents. This may offer the advantage of averting repeated drug administration while also avoiding the drug delivery complications posed by the blood-brain barrier. (Rosenstein, Science 235:772-774 (1987)).
As these critical factors have become recognized and optimized, intracerebral grafting has become a valid and reliable tool for neurobiologists in the study of CNS function and potentially for clinicians for the design of therapies of CNS disease, including brain tumors.
In parallel to the progress in neurobiology during the past several decades, advances in the understanding of molecular biology and the development of sophisticated molecular genetic tools have provided new insights into human disease in general. As a result, medical scientists and geneticists have developed a profound understanding of many human diseases at the biochemical and genetic levels. The normal and abnormal biochemical features of many human genetic diseases have become understood, the relevant genes have been isolated and characterized, and early model systems have been developed for the introduction of functional wild-type genes into mutant cells to correct a disease phenotype. (Anderson, Science 226:401-409 (1984)). The extension of this approach to whole animals, that is, the correction of a disease phenotype in vivo through the use of the functional gene as a pharmacologic agent, has come to be called xe2x80x9cgene therapyxe2x80x9d. (Friedmann et al., Science 175:949-955 (1972); Friedmann, Gene Therapy Fact and Fiction, Cold Spring Harbor Laboratory, New York (1983)). Gene therapy is based on the assumption that the correction of a disease phenotype can be accomplished either by modification of the expression of a resident mutant gene or the introduction of new genetic information into defective or damaged cells or organs in vivo.
Procedures for in vivo gene therapy have been described. See, e.g., Rosenberg et al., Science 242:1575-1578 (1988), and Wolff et al., Proc. Natl. Acad. Sci. USA 86:9011-9014 (1989), both incorporated herein by this reference, as well as co-pending U.S. patent application Ser. No. 07/285,196 by Gage, entitled xe2x80x9cMethod of Grafting Genetically Modified Cells to Treat Defects, Disease or Damage of the Central Nervous System,xe2x80x9d filed Dec. 15, 1988, and incorporated herein by this reference.
The anti-viral agents acyclovir (9-((2-hydroxyethoxy)methyl)guanine) and ganciclovir (9-((2-hydroxy-1-(hydroxymethyl)ethoxy)methyl) guanine) are efficient for preventing the replication of herpes virus, as the thymidine kinase coded for by the herpes virus genome and produced in cells infected by the herpes virus (HSV-TK) converts these drugs into intermediates capable of inhibiting DNA synthesis in vivo. Transfer of HSV-TK into tumor cells by retroviral vectors has been shown to mediate tumor regression from mouse sarcomas (Moolten and Wells, J. Natl. Cancer Inst., 82:297-300 (1990)) and to prevent growth of neoplastic BALB/c murine cell lines (Moolten, Cancer Res. 46:527-581 (1986)).
It would be advantageous to develop procedures for gene transfer via efficient vectors into cells followed by intracerebral grafting of genetically modified cells in vivo to treat brain tumors by introduction of therapeutic genes such as HSV-TK into the tumors.
The present invention provides methods for transferring therapeutic genes to brain tumor cells in order to kill the cells. In general, the method of the present invention comprises:
(1) introducing a retrovirus containing a selectable marker and at least one gene required for replication of the retrovirus into producer cells such that integration of the proviral DNA corresponding to the retrovirus into the genome of the producer cells results in the generation of a modified retrovirus wherein at least one of the genes required for replication of the retrovirus is replaced by the therapeutic gene or genes;
(2) selecting producer cells in which the modified retrovirus is incorporated as part of the genome of the producer cells;
(3) grafting the producer cells in proximity to the dividing tumor cells in order to infect the tumor cells with the modified retrovirus, thereby transferring the therapeutic gene or genes to the tumor cells; and
(4) killing the cells by administering a substance that is metabolized by the therapeutic gene or genes transferred to the tumor cells into a metabolite that kills the cells.
The tumor cells can be glioma cells. One of the genes transferred can be herpes simplex thymidine kinase (HSV-TK). The substance can be selected from the group consisting of: 9-((2-hydroxyethoxy)methyl) guanine and 9-((2-hydroxy-l(hydroxymethyl)ethoxy)methyl) guanine. A preferred retroviral vector for transferring HSV-TK can contain a NeoR gene and a control element selected from the group consisting of the thymidine kinase promoter, the SV40 early region promoter-enhancer, and the immunoglobulin heavy chain enhancer. The retrovirus can be derived from the Moloney murine leukemia virus.
More specifically, the invention comprises a method of preventing replication of tumor cells in vivo comprising:
(1) introducing a retroviral vector containing a selectable marker and the gene for herpes simplex thymidine kinase into producer cells such that integration of proviral DNA corresponding to the retroviral vector into the genome of the producer cell results in generation of a modified retrovirus wherein at least one of the genes required for replication of the retrovirus is replaced by the herpes simplex thymidine kinase gene;
(2) selecting producer cells carrying the herpes simplex thymidine kinase gene;
(3) grafting the producer cells carrying the modified retrovirus in proximity to the tumor cells in order to infect the tumor cells with the modified retrovirus, thereby transferring the herpes simplex thymidine kinase gene to the tumor cell; and
(4) administering an anti-cancer agent selected from the group consisting of: 9-((2-hydroxyethoxy)methyl) guanine and 9-((2-hydroxy-l(methyl)ethoxy)methyl)guanine such that the herpes simplex thymidine kinase gene metabolizes the anti-cancer agent into a metabolite that blocks replication of the tumor cells.